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Hello, everyone:

I just ran an experiment (flow cytometry, but would expect the same results
by confocal) staining T47D breast Ca cells with DiOC6(3) and PI to detect
apoptosis.  We had Jurkat cells stimulated with camptothecin for
controls.  Also,  m-chlorophenylhydrazone (CCCP) was used to trigger loss
of mitochondrial membrane potential in some of the controls.  The Jurkat
cell results were exactly as expected.

The results of the T47D cells, on the other hand, were very difficult to
interpret.  For those unfamiliar with this assay, the unperturbed cells
fall into the lower right-hand quadrant when DiOC is plotted on the x-axis
and PI is plotted on the Y-axis.  It should also be noted that the T47D
cells grow attached to the flask and were trypsinized ( and washed 2 times)
treatment with CCCP or staining with DiOC and PI.

The results for the T47D cells were as follows.  I gated on the viable cell
population based on fwd vs. side scatter.

1.  Untreated cells stained with both DiOC and PI were a little more
fluorescent for both stains than were Jurkat cells, so I adjusted the lines
that define the quadrants accordingly.  93% of the untreated cells were in
the lower rh quadrant.

2.  Treatment with CCCP, which should have moved the cells as a whole
across the line into the lower left hand quadrant, instead resulted in a
second DiOC peak forming to the right of the original (-) peak, both still
located for the most part in the lower rh quadrant.

3.  An attempt to induce apoptosis by depriving the cells of serum for 2
days resulted in the cells (DiOC/PI) being located in the lower rh quadrant
with a few cells leaking over into adjacent quadrants.  But, for the most
part, the cells remained located in the lower rh quadrant, but divided
between 2 peaks.

4.  Starvation plus CCCP not only resulted in a minor shift half-way across
the vertical quadrant line to the left, but also a shift completlely across
the horizontal quadrant line into the PI (+) space.  Remember that these
cells were gated on the live cell population by scatter.  Changing the
scatter gate to include only the "dead" cells resulted in a DiOC/PI  quad
plot that was virtually indistinguishable  from that generated through the
"live cell" gate.  This pattern was consistent for a duplicate set of samples.

Do any of you have any experience with this assay and/or with this cell
line?  Any explanation of the results?

Thanks in advance.

Rick Meister




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*  Richard K.
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